Title:

Display of Therapeutic Enzymes on Bacterial Spore Surface for Oral Delivery

Current effective and efficient methods of treating the severe metabolic disorder Phenylketonuria (PKU) are few and often highly uncomfortable for the afflicted person. PKU causes the buildup of phenylalanine in the blood for which the only available enzyme therapy is through weekly injections of phenylalanine ammonia-lyase (PAL) which reduces the levels of phenylalanine in the bloodstream. However, these regular injections can be painful and can come with potential side affects and therefore an oral delivery method is preferable. For this reason, bacterial spores were seen as a potential candidate to safely deliver PAL through the digestive tract and preserve it to provide long lasting treatment. When exposed to harsh environmental conditions several species of gram-positive bacteria will produce highly resilient spores. These spores are comprised of multiple dense protein layers which enable it to survive in unfavorable conditions such as intense heat or low pH. Due to these desirable properties, bacterial spores have been seen as a potential display system for enzymes and antigens. Experiments have already been performed which have demonstrated that foreign proteins can be fused to natural structural proteins in the spore allowing the foreign protein to be functionally displayed on the spore. In this work, PAL was fused to two potential structural protein candidates to be displayed on the spores of the bacteria Bacillus subtilis. Once displayed on the spore coat the efficiency of the fused PAL will be assayed via an enzyme activity assay.

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